Medium for growing human embryonic stem cells

• US 20050037492A1
• Filed: 09/24/2004
• Published: 02/17/2005
• Est. Priority Date: 01/11/2000
• Status: Active Grant

• Current Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Original Assignee: Geron Corporation
• Inventors: Li, Yan, Xu, Chunhui, Mandalam, Ramkumar
• Granted Patent: US 7,455,983 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• CPC Class Codes: C12N 2501/13 Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
• CPC Class Codes: C12N 2501/145 Thrombopoietin [TPO]
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/2306 Interleukin-6 (IL-6)
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2501/237 Oncostatin M [OSM]
• CPC Class Codes: C12N 2501/26 Flt-3 ligand (CD135L, flk-2 ligand)
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2502/99 genetically modified cells
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2510/04 Immortalised cells
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0619 : Neurons
• CPC Class Codes: C12N 5/0622 : Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells

Abstract

This disclosure provides an improved system for culturing human pluripotent stem cells. Traditionally, pluripotent stem cells are cultured on a layer of mouse embryonic fibroblast feeder cells to prevent them from differentiating. In the system described here, the role of feeder cells is replaced by defined components added to the culture environment that support rapid proliferation without differentiation. The medium contains an isotonic buffer, a blend of essential nutrients such as protein and lipids, and an effective growth factor or combination of factors that promote proliferation while inhibiting differentiation. Culturing human embryonic stem cells in fresh medium on an extracellular matrix according to this invention causes the cells to expand surprisingly rapidly, while retaining the ability to differentiate into cells representing all three embryonic germ layers. This new culture system allows for bulk proliferation of pPS cells for commercial production of important products for use in drug screening and human therapy.

Claims

1. A commercial product for culturing human embryonic stem (hES) cells in vitro such that they proliferate without differentiating, comprising:

a) a medium in which to culture the hES cells, comprising;

an isotonic buffer;

a protein nutrient, comprising serum, serum replacement, albumin, or essential and non-essential amino acids;

lipids, fatty acids, or cholesterol, either as artificial additives or as the HDL or LDL extract of serum; and

added fibroblast growth factor at a concentration of at least 40 ng/mL;

b) written information for growing hES cells in the culture medium.

2. The product of claim 1, wherein the medium also contains added nucleosides or nucleotides.

3. The product of claim 1, wherein the medium also contains added insulin.

4. The product of claim 1, wherein the medium also contains added transferrin.

5. The product of claim 1, wherein the fibroblast growth factor is recombinant basic human fibroblast growth factor.

6. The product of claim 1, wherein the medium contains between 40 and 200 ng/mL basic FGF.

7. The product of claim 1, wherein the medium contains at least 80 ng/mL basic FGF.

8. The product of claim 1, wherein the medium also contains added stem cell factor (SCF).

9. The product of claim 1, wherein the medium also contains added Flt3 ligand.

10. The product of claim 1, wherein all protein in the medium is human.

11. The product of claim 1, further comprising extracellular matrix components upon which to culture the hES cells.

12. The product of claim 9, wherein the extracellular matrix components comprise Matrigel®

basement membrane, laminin, fibronectin, proteoglycan, or entactin.

13. The product of claim 1, wherein the medium is provided in concentrated or powdered form.

17. A method for proliferating human embryonic stem (hES) cells in vitro, comprising obtaining the product of claim 1;

and culturing hES cells in the medium.

14. A commercial product for culturing human embryonic stem (hES) cells in vitro such that they proliferate without differentiating, comprising:

a) a concentrated solution that when diluted forms a medium in which to culture the hES cells, comprising;

an isotonic buffer;

a protein nutrient, comprising serum, serum replacement, albumin, or essential and non-essential amino acids;

lipids, fatty acids, or cholesterol, either as artificial additives or as the HDL or LDL extract of serum; and

added fibroblast growth factor at a concentration of at least 40 ng/mL;

b) written information for growing hES cells in the culture medium.

15. A commercial product for culturing human embryonic stem (hES) cells in vitro such that they proliferate without differentiating, comprising:

a) a dried powder that when dissolved forms a medium in which to culture the hES cells, comprising;

an isotonic buffer;

a protein nutrient, comprising serum, serum replacement, albumin, or essential and non-essential amino acids;

lipids, fatty acids, or cholesterol, either as artificial additives or as the HDL or LDL extract of serum; and

added fibroblast growth factor at a concentration of at least 40 ng/mL;

b) written information for growing hES cells in the culture medium.

16. A commercial product for culturing human embryonic stem (hES) cells in vitro such that they proliferate without differentiating, comprising:

a) separate components that when combined form a medium in which to culture the hES cells, comprising;

an isotonic buffer;

a protein nutrient, comprising serum, serum replacement, albumin, or essential and non-essential amino acids;

lipids, fatty acids, or cholesterol, either as artificial additives or as the HDL or LDL extract of serum; and

added fibroblast growth factor at a concentration of at least 40 ng/mL;

b) written information for growing hES cells in the culture medium.

18. A method of evaluating a medium designed for culturing human embryonic stem (hES) cells in vitro such that they proliferate without differentiating, comprising:

a) obtaining a medium comprising;

an isotonic buffer;

a protein nutrient, comprising serum, serum replacement, albumin, or essential and non-essential amino acids;

lipids, fatty acids, or cholesterol, either as artificial additives or as the HDL or LDL extract of serum; and

added fibroblast growth factor at a concentration of at least 40 ng/mL;

b) culturing hES cells in the medium through at least four passages; and

c) determining whether the hES cells cultured in the medium have proliferated without differentiation.

19. A culture environment for growing human embryonic stem (hES) cells in vitro such that they proliferate without differentiating, comprising:

a culture device;

an extracellular matrix component;

an isotonic buffer;

a protein nutrient, comprising serum, serum replacement, albumin, or essential and non-essential amino acids;

lipids, fatty acids, or cholesterol, either as artificial additives or as the HDL or LDL extract of serum;

added fibroblast growth factor at a concentration of at least 40 ng/mL.

20. A culture of human embryonic stem (hES) cells that proliferate without differentiating, comprising hES cells growing in the culture environment of claim 19.